Polystyrene culture flasks

SH-SY5Y human neuroblastoma-derived cell lines used in this work were cultured in polystyrene culture flasks (Corning) at 37°C with 5% CO2 in DMEM (Gibco, Invitrogen, 11965092) medium supplemented with 10% Fetal Calf Serum (Gibco, Invitrogen, 10500056), 1 mM Sodium pyruvate (Gibco, Invitrogen, 11360070) and antibiotics (50 U/ml of penicillin, streptomycin and nystatin).
HEK293 cells were grown in the same conditions as above. The SHSY5Y human neuroblastoma FD model (Elp1 KD) and PLKO vector control were generated in our laboratory as previously described (Cohen-kupiec et al., 2010 (link)). The SHSY5Y human neuroblastoma Tau knockdown model (Tau KD) and control were kindly provided by Dr. Paganetti’s Laboratory for Biomedical Neurosciences (LBN) Neurocenter of Southern Switzerland (NSI) (Sola et al., 2020 (link)).

NB-TA-MSC (n = 6) were derived from NB biopsies and termed as 2ZC, FA, DI, BU, PGE and CO. NB primary cultures were isolated after tumor tissue mechanical dissociation, and collagenase type II treatment as previously described [20 (link)]. The cell suspensions were initially collected and plated in polystyrene culture flasks (Corning Costar, Corning, NY, USA) and isolated based on their ability to adhere to plastic as fibroblast-like cells after being in culture at 37  °C in a humidified atmosphere with 5% CO₂ in D-MEM + GlutaMAX (Gibco, Billings, MT, USA) supplemented with 10% of fetal bovine serum (FBS; Euroclone, Milan, Italy), 50 mg/mL gentamicin and 1% penicillin. Thereafter, NB-TA-MSC were expanded in culture up to senescence at 37 °C in a humidified atmosphere with 5% CO₂ in the MesenPRO RS™ Medium (Gibco, Billings, MT, USA, 12746012), supplemented with 2 mM L-Glutamine (Gibco, Billings, MT, USA, A29168-01) according to previously described procedures [7 (link)]. This medium is specifically formulated to promote the growth of human mesenchymal stem cells (MSCs) for multiple passages while preserving their multi-potential phenotype. In addition, the phenotypic molecular and functional properties of the above-mentioned TA-MSC have been thoroughly characterized elsewhere [20 (link)].

MSCs were isolated after tumor tissue mechanical dissociation and collagenase type II treatment as previously described [16 (link)]. The cell suspension was then collected and cultured following standard expansion procedure for bone-marrow MSC (BM-MSCs). Briefly, cells were plated in polystyrene culture flasks (Corning Costar, Corning, NY, USA) at a density of 160,000/cm² in D-MEM + GlutaMAX (Gibco) supplemented with 10% FBS (Euroclone), 50 mg/mL gentamicin and 1% penicillin.
NB-MSCs were isolated based on their ability to adhere to plastic as fibroblast-like cells, after being in culture at 37 °C in a humidified atmosphere with 5% CO₂ and culture medium replacement twice a week. At ≥80% confluence, NB-MSCs were harvested by Trypsin EDTA (Euroclone), replated for expansion at a density of 4000 cells/cm². Previously expanded and characterized BM-MSCs were used as controls for the experiments described in this study.

BM cells from both patients and HDs were collected as residual samples of the CD138 immuno-magnetic depletion performed for another ongoing research program. We conducted preliminary experiments assessing both intact and CD138 cell-depleted BMs, indicating that CD138 depletion did not alter either the AL- or HD-MSC expansion/differentiation capacity and their in vitro survival (data not shown). Therefore, we performed MSC isolation and expansion only from CD138neg BM-MNCs, following a standard in vitro culture procedure [23 (link),24 (link)]. Briefly, MNCs were obtained from BM aspirates by density gradient separation and plated in polystyrene culture flasks (Corning Costar, Corning, NY, USA) at a density of 160,000/cm² in DMEM + GlutaMAX (Gibco, Life Technologies, Milan, Italy) supplemented with 10% fetal calf serum (FCS) (Euroclone, Milan, Italy) (MSC-complete medium) and incubated at 37 °C, 5% CO₂. This step was defined as P0 and culture medium was replaced twice a week. MSCs were harvested by Trypsin EDTA (Euroclone) at ≥80% confluence and re-plated for expansion at a density of 4000 cells/cm².